During the past year, this laboratory continued studies on the mechanism of HIV entry, and on the development of novel protective and treatment strategies based on the molecules involved in entry. 1) Correlation between coreceptor usage and antibody neutralization sensitivity for different HIV-1 isolates. Using MAb D19 directled against a highly conserved epitope within the V3 loop of gp120, we found that for R5 isolates, the epitope is masked in the native trimer and becomes exposed only upon CD4 binding. By contrast, for X4 and R5X4 isolates, the epitope is exposed even in the native trimer. For all isolates, the epitope is exposed on the soluble gp120 subunit. These results demonstrate a correlation between coreceptor usage phenotype of different HIV-1 isolates, and aspects of the quaternary structure that influence exposure of an epitope critical for function. 2) Novel anti-HIV agents based on HIV Env/receptor interactions. a) sCD4-17b, a potent HIV-1 neutralizing agent designed in this laboratory. sCD4-17b is a recombinant chimeric protein containing soluble CD4 attached via a long flexible polypeptide linker to a single chain antibody that binds to a CD4-induced epitope of gp120 involved in binding to coreceptor. While the protein neutralizes some HIV-1 strains with extremely high potency, other strains appear resistant, despite the known presence of binding sites for CD4 and 17b. We hypothesize that the resistance of these isolates reflects structural features of the corresponding gp120 molecules (V1/V2 and V3 loops, glycosylation) that render the original linker (35 residues) insufficiently long to enable simultaneous binding of the CD4 and 17b moieties. We have engineered sCD4-17b variants with longer linker lengths, and prepared corresponding recombinant baculoviruses for high level expression. Large amounts of these proteins have been prepared, and they will be tested for from breadth of neutralization. We have also continued efforts to express sCD4-17b in Lactobacillus, with the idea of colonizing the vaginal tract to provide a means for protection against sexual transmission that is durable, economically feasible, and controlled by women. b) Anti-HIV immunotoxin. NIH has licensed 3B3-PE38 to IVAX, to produce clinical grade material for Phase I trials.